1. Field of the Invention
This invention concerns a method for diluting blood, blood diluent, and antimicrobial agents and reagents, especially suitable for use in enumeration and sizing of blood cells, including white blood cell subpopulations.
2. Description of the Prior Art
Analytical data of blood are important as an index of health and well being. The analysis of blood cell populations provides useful information for diagnosis and treatment, since they show a rapid response to variations in medical condition.
Most analytical methods rely on the fundamental cell property of volume regulation. Each type of cell in the circulating blood has its own characteristic volume, ranging from as small as 2 cubic microns for platelets to over 450 cubic microns for polymorphonuclear cells. Automated, blood-analysis instruments have been developed to accomplish the measurement of blood cells and related components with simplicity and rapidity. Such measurements include white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hematocrit (HCT), hemoglobin (HGB), the mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC), as well as WBC subpopulations such as lymphocytes, monocytes, and granulocytes.
Before making blood cells counts by means of an automated, blood-analysis instrument, the blood is diluted to a certain concentration. The diluent comprises a stable water solution of chemical salts providing an electrolytic solution capable of conducting current to which a blood sample can be added so as to dilute the red blood cells, white blood cells, platelets and other blood components and enable the desired parameters of these blood components to be measured, counted and evaluated. See, for example, U.S. Pat. Nos. 4,213,876, 4,244,837, 4,521,518 and 4,745,071.
In analyzing blood by an automated, blood-analysis instrument or other analytical method (including manual microscopic evaluations), it is essential that the diluent not adversely affect the chemical and physical integrity of the blood cells during the analysis. For instance, if the blood diluent is not isotonic and osmotically balanced with respect to the blood, the blood cells may shrink or expand. Importantly, The diluent should not adversely affect the blood analysis itself.
The diluent must also be free from particles which may interfere with the analysis of the blood. While the diluent is normally filtered to remove particles larger than 0.2 micron in diameter at the time of its manufacture, the diluent may be susceptible to the support of microorganism growth after the manufacture and packaging of the diluent. The presence of microorganisms may result in inaccurate and non-reproducible results.
It is a common practice to include a antimicrobial agent in blood diluents to retard the growth of microorganisms. The antimicrobial agent, as with other components of the diluent, must not adversely affect the blood cells or adversely affect or interfere with the analyses. Thus, while antimicrobial agents in blood diluents are desireable, caution must be observed in their selection and use.
An antimicrobial agent which has been employed for many years in blood diluents is sodium azide. Its use has not, however, been without significant problems. First, the presence of sodium azide has been found to influence the formation of cyanmethemoglobin, a chromogen formed for determining hemoglobin in a blood sample. For instance, without sodium azide in the diluent, the hemoglobin as determined photometrically may be significantly different than that with the azide present. Second, aqueous solutions of sodium azide are highly toxic (toxicity level of 6 on a scale of 1 to 6). See Gosselin et al. (eds.), Clinical Toxicology of Commercial Products, The Williams and Wilkins Company, Baltimore, 5th Edition, 1984, (Section IID, position 111). Finally, the disposal of sodium azide through copper or lead-containing plumbing systems may result in the formation of heavy metal azides and their buildup over extended period of time.
Despite the need for alternatives to sodium azide as an antimicrobial agent in blood diluents, few alternatives have been proposed. An attempt has been made to substitute other preservatives for sodium azide and an example of this is shown in U.S. Pat. Nos. 3,962,125 and 4,102,810 where 2-phenoxyethanol is used as an antimicrobial agent.
There are disadvantages to the use of phenoxyethanol as well. First, phenoxyethanol is a rather weak antimicrobial agent. It has been found to be more effective when used in conjunction with other preservatives, such as the hydroxybenzoates. Martindale, The Extra Pharmacopoeia (27th edition, The Pharmaceutical Press, London, 1977, page 1281). Second, while less toxic than sodium azide, phenoxyethanol is still toxic (toxicity level of 4 on a scale of 1 to 6). See Gosselin et al., supra (Section IID, position 463). Finally, in U.S. Pat. Nos. 3,962,125 and 4,102,810, sodium fluoride is essentially included along with phenoxyethanol in the proposed diluent to enhance hemoglobin chromogen formation. Sodium fluoride is only slightly less toxic than sodium azide (toxicity level of 4-5). See Gosselin et al., supra (Section IID, position 100).
A further attempt to substitute other preservatives for sodium azide can be found in U.S. Pat. No. 4,248,634, in which sodium dehydroacetate is used as an antimicrobial agent. Sodium dehydroacetate, a fungicide, is toxic at high doses but is less toxic than sodium azide, sodium fluoride, and phenoxyethanol (toxicity level of 3). See Gosselin et al., supra (Section IID, position 1187).
One object of the present invention is to provide an antimicrobial agent for a blood diluent that does not interfere with the blood analysis.
Another object of the present invention is to provide an antimicrobial agent for a blood diluent that is potent but relatively non-toxic.
Still another object of the present invention is to provide an antimicrobial agent that is potent but inexpensive.
Objects and advantages other than those above set forth will be apparent from the following description when read in connection with the accompanying drawings.